Swine influenza is one of the economically significant acute respiratory diseases in pigs of all ages throughout the world since it was initially recognized in the early 1900's (Shope, J. Exp. Med. 54:373-385 (1931)). Occasionally, the disease is also presented as a reproductive problem in breeding animals at any time of gestation. Because of its economic significance, vaccination of gilts and sows using inactivated vaccines has been commonly practiced to prevent reproductive loss in breeding animals and productivity loss in young pigs due to respiratory disease.
The disease is caused by swine influenza virus (SIV), which is a member of the genus Influenzavirus in the family Orthomyxoviridae (Esterday et al., “Swine Influenza.” In: Diseases of Swine. Iowa State University Press, Ames, Iowa, Straw et al., eds., 1999, pp. 277-290). SIV contains 8 segmented RNA molecules, each of which encodes for 4 structural proteins (i.e., hemagglutinin (H), neuraminidase (N), nucleoprotein (NP), and matrix (M1 and M2)), and nonstructural proteins (three polymerases (PA, PB1 and PB2) and non-structural (NS) proteins NS1 and NS2). The NP and M1 proteins are internal structural proteins and serve as antigenic determinants for the type (i.e., A, B, or C) of influenza virus as these proteins are antigenically most conserved among the same type. The M2 is transcribed from the M segment through gene splicing, acts as an ion channel, and favors viral entry (Hay, Semin. Virol. 3:21-30 (1992)). The H and N proteins are surface proteins specific for subtypes and play a critical role in viral entry to and release from target cells, respectively. To date, three subtypes of influenza A viruses, such as H1N1, H1N2, H3N1 and H3N2, have been consistently implicated in swine influenza, although 15 and 9 different H and N subtypes, respectively, are known to exist among avian and mammalian influenza viruses (Esterday et al. (1999), supra). The NS1 protein is expressed only during the natural infection and has shown a role in down-regulating the host innate immune response (Palese et al., Arch. Virol. Suppl. 15: 131-138 (1999); and Wang et al., J. Virol. 74: 11566-11573 (2000)), while NS2 protein, which is expressed from the NS1 gene through gene splicing, functions as a Nuclear Export Protein (NEP) for translocation of ribonucleoptroteins (RNPs); (Hilleman, Vaccine 20: 3068-3087 (2002); and O'Neill et al., EMBO J. 17: 288-296 (1998)).
A definitive diagnosis of swine influenza requires the detection of virus or viral antigen in tissues and/or secretions of clinically affected animals. Nonetheless, serological tests, such as hemagglutination inhibition (HI) test, serum-virus neutralization test, indirect fluorescent antibody test, or enzyme-linked immunosorbent assay (ELISA), are often employed to detect animals that have been exposed to the virus because the disease has a very short course and because the causative agent becomes undetectable in infected animals quickly after infection (Yoon et al., Swine Influenza Virus: Evolution, Epidemiology and Diagnosis. In: Trends in Emerging Viral Infections of Swine, Iowa State University Press, Ames, Iowa. Morella et al., eds., 2002, pp. 23-28). Serology is also used to assess the immune status of pigs at various stages within an operation so that the level of herd immunity or timing of vaccination can be determined. The diagnostic value of serologic data, however, is frequently confounded by several factors, such as antigenic difference between the virus that induced the antibody and the virus that was used in the test (Long et al., J. Vet. Diagn. Invest. 16: 264-270 (2004)), and the difficulty in differentiating whether detected antibody is of maternal origin, or was induced by infection or vaccination (Renshaw, Am. J. Vet. Res. 36: 5-13 (1975)).
Until the early 1990's, only classical H1N1 had circulated in U.S. swine populations. Moreover, the HI assay has been most commonly used for serodiagnosis of SIV infection in veterinary diagnostic laboratories in North America because: a) it is relatively inexpensive; b) the antigenicity of SIV in the U.S. swine population has been very stable with respect to the subtype of the implicating virus; and c) results show a relatively good prediction for the immune protection of pigs from subsequent infection. However, such utility and reliability of this subtype-specific assay was recently diminished due to the emergence of new subtypes and new reassortants (e.g., H3N2, H1N2, H3N1, and H9N2; etc.) in U.S. pig populations, as well as progressive antigenic drift within the same subtype (Webby et al., Virus Res. 103: 67-73 (2004); and Webster et al., Microbiol. Rev. 56: 152-179 (1992)).
Accordingly, there remains a need for a subtype-unrestricted universal serologic assay to detect pigs exposed to SIV and, at the same time, preferably differentiate exposed animals from vaccinated animals. It is an object of the present invention to provide materials and a method for such an assay. This and other objects and advantages of the present invention, as well as additional inventive features, will become readily apparent from the detailed description provided herein.